The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.
Sphingolipids, a class of compounds defined by their common 18 carbon amino alcohol backbones, mediate cell-cell and cell-substratum interactions, modulate the behavior of cellular proteins and receptors, and participate in signal transduction. They are therefore important regulators of cell growth, differentiation and survival. The sphingolipids are synthesised de novo from palmitoyl-CoA and serine via a pathway whereby the carbon backbone, alcohol and amino groups are modified to form the various bioactive compounds, such as ceramide, sphingosine and sphingosine-1-phosphate (Scheme 1). Perturbations in the sphingolipid biosynthetic pathway are implicated in many physiological and pathophysiological processes, including cancer, diabetes, inflammation, and Alzheimer's disease.

One of the most studied sphingolipids is sphingosine-1-phosphate (S1P) which is formed by the phosphorylation of sphingosine (Sph) by two kinases: sphingosine kinase 1 (SphK1), located mainly in the cytosol, and sphingosine kinase 2 (SphK2), located in several intracellular compartments. S1P levels are controlled by numerous factors, including the SphKs, and by enzymes that degrade S1P (see M Maceyka et al, Trends in Cell Biology, 2012, 22, 50-60 and references cited therein).
S1P plays a key role in cancer progression, regulating cell growth, suppression of apoptosis, tumour angiogenesis, metastasis and chemoresistance, and is capable of up-regulating a variety of pro-survival pathways and down-regulating apoptotic pathways. Accordingly, an increase in SphK1 expression and activity leads to a marked shift in the Sph:S1P ratio, in favour of S1P. This in turn triggers a series of pro-survival pathways (glycolysis, angiogenesis, metastasis etc) and cell growth and down regulates apoptotic pathways, promoting the survival and spread of cancer cells. Numerous model studies indicate that over-expression of SphK1 promotes tumour growth whereas inhibition reduces tumour growth, angiogenesis and chemoresistance (resistance is often associated with sustained SphK1 expression). Down regulation of SphK2 has also been shown to inhibit cancer cell growth and enhance chemotherapy induced apoptosis. (M Maceyka et al, supra, and S Pyne et al, Cancer Res. 2011, 71, 6576-82, and references cited therein).
S1P also plays important roles in fibrotic disease. Fibrosis is a pathologic condition involving aberrant and uncontrolled extracellular matrix production by the connective tissue as a result of injury or disease, leading to excessive scarring. This leads to increasing tissue dysfunction and, ultimately, organ failure. Fibrosis is a key cause of heart, lung, liver and kidney failure in diseases such as heart attack, diabetic nephropathy, idiopathic pulmonary fibrosis and cirrhosis of the liver. Heightened levels of S1P have been detected in fibrotic tissue and S1P has been shown to be a promoter of a number of the cellular processes that contribute to fibrosis: cell differentiation into fibroblasts and myofibroblasts (scar forming cells), extracellular matrix (ECM) production by myofibroblasts, hypertrophy and mast cell activation (Takuwu N. et al. Sphingosine-1-phosphate in cardiac fibrosis. Inflammation and Regeneration, 2013, 33(2), 96-108).
Asthma is a chronic inflammatory disorder leading to wheezing, breathlessness and coughing and its incidence in developed nations is increasing. Studies have demonstrated the key role of the SphK1 and 2/S1P pathway in the development of asthma by regulating pro-inflammatory responses where blockade of SphK1/2 activity has been shown to supress airway inflammation (W-Q. Lai, et al, Bioscience Reports, 2011, 31, 145-50, and references cited therein).
Evidence also implicates the role of S1P in both neuropathic and nociceptive pain in diverse etiologies and regulation of the activity of SphK1 and/or SphK2 has been suggested as offering potential for the development of analgesics (D. Salvemini et al, Trends in Pharmacological Sciences, 2013, 34, 110-118, and references cited therein).
Small molecules inhibitors of SphK1 and SphK2 can bind to either the substrate (sphingosine) binding domain or the ATP binding domain (C. Loveridge et al. J. Biol. Chem. 2010, 285, 38891; K. G. Lim et al. J. Biol. Chem. 2011, 286, 18633). A third site on SphK1 has also been identified from competitive binding studies and is termed the allosteric site. In extracellular studies on SphK1 activity, compounds that bind to the allosteric site may either enhance or inhibit enzymatic activity. In cells, however, these compounds may also promote to polyubiquination and proteasomal degradation of the protein (SphK1). It has been proposed that this allosteric site may indeed be an autoregulatory domain where S1P binds to down-regulate SphK1 through both enzymatic inhibition and down-expression (proteasomal degradation). Exogenous ligands that bind to this site may also block the enzyme through both processes (enzymatic inhibition and degradation) or, alternatively, may block S1P binding and fix the enzyme in an active conformation, promoting S1P production. Thus, exogenous allosteric binders may either act as allosteric blockers or enhancers of SphK1 activity.
Another enzyme in the sphingolipid signalling pathway, dihydroceramide desaturase-1 (Des1), has also been implicated in disease. Des1 is active in an earlier stage of the biosynthetic pathway and mediates the conversion of dihydroceramide (dhCer) (e.g. n=16) to ceramide (Cer) (e.g. n=16) by the introduction of the 4,5-double bond into the carbon backbone (see Scheme 1, supra). Since both dhCer and Cer are metabolised into other sphingolipids by the same enzymes, Des1 is responsible for the overall relative levels of all dihydrosphingolipids compared to their Δ4-unsaturated counterparts. The accumulation of dhCer and other dihydrosphingolipids that results from blocking Des1 has been shown to have therapeutic potential in cancer, metabolic disease and viral and bacterial infection (Gagliostro V et al. Prog. Lipid Res. 2012, 51. 82-94). The anticancer effects of Des1 inhibition are linked to a combination of apoptotic and autophagic cancer cell death. While the mechanistic details of this remain to be discerned, it is possible that by inhibiting Des1 the downstream S1P is decreased and the upstream dihydrosphingosine-1-phosphate (dhS1P) is increased. Recent studies have shown that dhS1P has opposing effects to S1P in cancer and fibrosis (Bu, S. et al. J. Biol. Chem. 2008, 283(28), 19563-19602). DhS1P is able to block the activation of fibroblast that is mediated by S1P and other growth factors such as transforming growth factor-β (TGFβ). Also, in cancer, injections of S1P and dhS1P have opposing effects on tumour growth in xenograft models, S1P promotes growth and dhS1P suppresses it (Barth B. M. et al. ACS Nano. 2013, 7, 2132-2144).
Notwithstanding their importance in cellular function and survival, the study of these sphingolipid enzymes has thus far been limited at least in part due to the paucity of suitable exogenous agents which target or interact with them. Therefore, a need exists for the identification of new agents which can interact with SphK1 and/or SphK2 and/or Des1.